Antibiotics



ANTIBIOTICS Winston Kennay Anslow, Slough, and George Desmond Wilkin, Uxbridge, England, assignors to Glaxo Laboratories Limited, Greenford, Middlesex, England, a British company No Drawing. Filed Jan. 10, 1957, Ser. No. 633,380

11 Claims. (Cl. 195- 80) This invention is concerned with improvements in or relating to antibiotics and is more particularly concerned with improved methods for the production of the antibiotic described in copending application No. 602,634, filed August 7, 1956, and now abandoned in favor of continuation-impart application 767,343, filed October 15, 1958, and there designated E. 129.

The antibiotic E. 129, as disclosed in said application has been obtained by the aerobic cultivation on or in a suitable nutrient medium of an organism discovered by us and identified as belonging to the genus Streptomyces. This organism, which was isolated from a soil sample collected near Jericho in Jordan, was originally designated as Streptomyces E. 129, but is now named Streptomyces ostreogriseus.

Cultures of the organism have been deposited with the National Collection of Industrial Bacteria (N.C.I.B.) at the Department of Scientific and Industrial Research, Teddington, England, under number N.C.I.B. 8792 and also in the culture collection of the Northern Regional Research Laboratories (N.R.R.L.) at Peoria, Illinois, United States of America, under number N.R.R.L. 2558.

We have now found that improved results, particularly improved titres, are obtained in the production of E. 129 by culturing an E. 129 producing organism, particularly Streptomyces ostreogriseus on or in a suitable medium, if the medium contains available iron in a concentration Within the range of from 130 135 parts per million (ppm) and desirably within the range of from 35-55 p.p.m. A concentration of iron above 135 p.p.m. begins to show substantially inhibiting effect on the organism.

As previously stated in the said copending application it is preferred to produce E. 129 by the submerged fermentation of an E. 129-producing organism in a nutriatent ent medium containing a complex organic source of nitrogen such as oatmeal, soya bean meal, corn steep liquor and the like, a carbohydrate and mineral salts. As is known organic materials such as the above mentioned sources of nitrogen often contain trace quantities of iron, and this should be allowed for in computing the quantity of iron to be added to the medium for the purpose of the present invention. It is the case however that many of the more important complex organic sources of nitrogen used in the culture of microorganisms contain substantially the same amount of iron, i.e. from 10-20 p.p.m., so that an amount of additional iron of at least 25 p.p.m. and not more than 115 p.p.m. will usually be satisfactory. If an organic material with a greatly different iron content is used the amount of added iron must of course be correspondingly adjusted.

The nature of the source of the added iron does not appear to be important provided it is soluble in the fermentation medium and is substantially non-toxic to the organism and compatible with the resultant antibiotic; iron in both the ferrous and ferric states may be used. We have found that suitable sources of iron are ferric citrate, ferric chloride and ferrous ammonium sulphate.

-It is known that limestone, (chalk) chelates with iron. In certain of the media which we have used for the production of E. 129 we have used limestone as an ingredient together with iron and, in spite of the known chelating properties of limestone with iron, an increase in titre was obtained. V

It has also been found that an improved-titre in the production of E. -129 by fermentation is obtained when com steep liquor is used in a medium also containing oatmeal and a source of carbohydrate. A particularly suitable quantity of corn steep liquor is 04-09% (by weight of solids) of the weight of the medium, preferably an amount of 0.7% by weight of solids. The preferred amounts of oatmeal are within the range 15%, advantageously 2.5%.

The ratio of corn steep liquor to soluble carbohydrates is also important in obtaining good titres. Thus-while in the absence of corn steep liquor no appreciable difference in titre is obtained using two media which'contain, inter alia 0.5% and 1.5% by weight respectively of glucose-if corn steep liquor, say 0.7% by weight of solids, is added to each medium not only is an increase in titre of each obtained, but also an enhance titre is obtained with the medium containing 1.5 glucose compared with that containing only 0.5% glucose. The eifect of the corn steep liquor and source of carbohydrate would therefore appear to be synergistic.

In general we have found it preferable to have present 1.5% of soluble carbohydrate, especially glucose, when corn steep liquor is used.

A medium which we have found to be very suitable for use in the production of E. 129 is one containing oatmeal flour (preferably 2.5%), limestone (preferably 2.0%) and glucose (preferably 1.5 adjusted to a pH of 6.8-7.0 with e.g. hydrochloric acid or NaOH and to which has been added corn steep liquor in anamount as set out above and preferablyalso containing added iron in an amount as set out above (preferably '30 p.p.m.).

In order that the invention may be well understood the following examples will now be given by way of illustration only, the parts being by weight.

A. EXAMPLES SHOWING THE EFFECT OF IRON Example 1 Glucose (sterilized separately) 1.5

(pH adjusted to pH 6.8-7.0 with sodium hydroxide).

To one set of fermenters was added 60 parts per'mil lion of iron as ferric citrate. lated with 2% of a vegetative culture of Streptomyces E. 129 (grown on the above oatmeal medium without the addition of corn steep liquor and containing only 0.5% glucose) and fermented at 27 C. with an air flow.

of 3 litres per minute. Assays against Sarcina lutea gave the followingresultsinu./1nl.:' I

Hours Controls Plus-60 (no iron) p.p.m. Fe

Each fermenter was inocu-l Example 2 The effect of iron was further explored using the same medium and conditions as in Example 1 but with levels of 30, 60 and 120 parts per million of iron in the form of ferric citrate. The average titres (three fermenters in each set) were as follows: r r

Example 2 was repeated using lower levels of iron, viz. 15, 30 and 45 parts per million, with the following results (averages of three fermenters):

Hours Controls Plus Plus 30 Plus 45 (no iron) p.p.m. Fe p.p.m. Fe p.p.m. Fe

Example 4 To ensure that different sources of iron were equally beneficial three sets of four fermenters were prepared as in the Examples 1-3 but all with the addition of 30 parts per million of iron, one set using ferric citrate as before, another set using ferric chloride and the third set using ferrous ammonium sulphate. The following Example 6 To determine the best level of corn steep liquor four sets of three 5-litre fermenters were prepared, containing the basal oatmeal medium with 1.5% glucose. To one set was added 0.45% corn steep solids, to the second 0.7%, to the third 0.9% and to the fourth 1.1%. The fermenters were inoculated and fermented as before. The results are given in the table below, each figure being the average of three fermenters:

Corn steep liquor solids Hours It is evident that 1.1% corn steep solids has a deleterious eifect on titre and that 0.9% is a reasonable top limit.

C. EXAMPLE SHOWING THE EFFECT OF GLUCOSE Example 7 The effect of additional glucose was next determined. 5-litre fermenters containing the basal oatmeal medium with glucose at 0.5% and 1.5% levels and fermenters containing the basal oatmeal medium plus 0.7% corn steep solids with glucose at 0.5% and 1.5 levels were prepared, inoculated and fermented as in Example 5, with the following results in u./ml.:

results were obtained, the figures being the average of OatmealMedium outmefllMedmm +01% each Set: Corn Steep Liquor solids 30 p.p.m. 30 p.p.m. 30 p.p.m. Hours Glucose Glucose Glucose Glucose, ironas iron as iron as I Hours ferric lflelerrle1 ferrous citrate c ori e ammonium sulphate It is clear that any of these salts can be used, It will be seen that the use of 1.5% glucose in the B. EXAMPLES SHOWING THE EFFECT OF CORN STEEP LIQUOR Example 5 Three 5-litre fermenters were batched with three litre quantities of the following medium, the glucose portion being sterilised separately:

Percent Oatmeal 2.5 Limestone 2.0 Glucose 0.5

Corn steep liquor solids Hours It will be seen that the lowest level of corn steep liquor gave no increase over the usual titre of 200-300 u./ml. which experience has shown to be obtainable on the basal medium, whereas 0.46% and 0.70% corn steep liquor gave substantial increases.

basal oatmeal medium has no beneficial effect over 0.5%, whereas a substantial increase in titre is obtained by the use of 1.5% glucose when 0.7% corn steep liquor solids are included.

We claim:

1. A process for the production of an antibiotic-containing medium of improved titre which comprises culturing Streptomyces ostreogriseus N.R.R.L. No. 2558 under submerged aerobic conditions in a liquid culture medium containing from 30-135 parts per million of available iron.

2. A process as claimed in claim 1 in which said medium contains from 35-55 parts per million of available 11011.

3. A process for the production of an antibiotic-com taining medium of improved titre which comprises culturing Strepzomyces ostreogriseus N.R.R.L. No. 2558 under submerged aerobic conditions in a liquid culture medium containing a complex organic source of nitrogen, a carbohydrate and nutrient mineral salts and added assimilable iron in an amount of from 25-115 parts per million of said medium.

4. A process as claimed in claim 3 in which said medium contains as an added source of iron at least one iron salt selected from the group consisting of ferric 6. A process as claimed in claim 3 in which said me-' dium comprises glucose as carbohydrate.

7. A process for the production of an antibiotic-containing medium of improved titre which comprises culturing Streptomyces ostreogriseus N.R.R.L. No. 2558 under submerged aerobic conditions in a liquid culture medium containing a source of carbohydrate and a source of nitrogen comprising from 0.4 to 0.9% by weight of corn steep liquor solids and from 1 to 5% by weight of oatmeal.

8. The process as claimed in claim 7 in which said medium contains from 05-15% by weight of carbohydrate.

9. The process as claimed in claim 7 in which said culture medium also contains added assimilable iron in the amount of from 25-115 p.p.m. of said medium.

10. A process for the production of an antibiotic-containing medium of improved titre which comprises culturing Streptomyces ostreogriseus N.R.R.L. No. 255 8 under submerged aerobic conditions in a liquid culture medium containing about 2.5% by weight of oatmeal flour, about 0.7% by weight of corn steep liquor solids, about 2.0% by weight of limestone, and about 1.5% by weight of glucose.

11. The process as claimed in claim 10 in which said culture medium contains about 30 p.p.m. of added assimilable iron.

5 References Cited in the file of this patent UNITED STATES PATENTS 2,787,580 Sobin et al. Apr. 2, 1957 10 FOREIGN PATENTS 776,035 Great Britain May 29, 1957 793,797 Great Britain Apr. 23, 1957 OTHER REFERENCES 15 Foster: Botanical Review, vol. 5, No. 4, 1939, The

Heavy Metal Nutrition of Fungi, pp. 224 to 227.

Charney et al.: Antibiotics Annual, 1953-1954, pp. 171-173.

Celmer et al.: Antiobiotics Annual, 1955-1956, pp. 20 439-452 (Nov. 2, 3 and 4, 1955). 

1. A PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC-CONTAINING MEDIUM OF IMPROVED TITRE WHICH COMPRISES CULTURING STREPTOMYCES OSTREOGRISEUS N.R.R.L. NO. 2558 UNDER SUBMERGED AEROBIC CONDITIONS IN A LIQUID CULTURE MEDIUM CONTAINING FROM 30-135 PARTS PER MILLION OF AVAILABLE IRON. 